Native-state method and system for determining viability and proliferative capacity of tissues in vitro

ABSTRACT

The present invention relates to a method of using an in vitro culture system to measure the cellular proliferative capacity cellular viability of human tissues, particularlytumor tissues. The invention also describes the use of the method to evaluate the effectiveness of an antineoplastic drug in inhibiting tumor cell proliferation of viability.

This application is the national phase under 35 U.S.C. §371 of PCTapplication WO 95/01455, filed Jun. 29, 1994, which claims priority fromU.S. application Ser. No. 08/084,402 filed 29 Jun. 1993 and nowabandoned. This application is a continuation-in-part of U.S. Ser. No.08/084,402, filed Jun. 29, 1993, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/965,602, filed Oct. 22, 1992,now abandoned, which is a file wrapper continuation of U.S. Ser. No.07/326,286, filed Mar. 20, 1989.

DESCRIPTION

1. Technical Field

The present invention relates to a method of using an in vitro culturesystem to measure the cell proliferation and cell viability of humantissues, particularly tumor tissues, and thereby to measure the efficacyof anti-neoplastic drugs upon the proliferation and viability of thecultured cells.

2. Background

Cancer is a disease involving inappropriate cell division. A realisticmodel is greatly needed to understand the biology of alteredproliferation in cancer as compared to normal tissue and to useinformation on proliferation capacity as a basis of cancer prognosis andtreatment.

Measurements of proliferation capacity of tumors currently are obtainedby thymidine-labeling index (TLI), by flow cytometric measurements ofcells presumed to be in S phase, or by measuring a nuclear antigen,Ki-67, found in at least certain proliferating cell types. Meyer et al.,Breast Cancer Rest. Treat., 4: 79-88 (1984); McDivitt et al., Cancer,57: 269-76 (1986); and McGurrin et al., Cancer, 59: 1744-50 (1987).Whichever method is used, the results obtained show that the higher theS-phase fraction is, the poorer the prognosis. Clinical studiesutilizing the TLI procedure have been successful in identifying anddetermining therapy of a subgroup of lymph node-negative women withbreast cancer having a 48% relapse rate. Meyer et al., Cancer, 51:1879-86 (1983). There is therefore great potential value for cancerprognosis, therapy, and biology in determining the proliferativecapacity of tumors.

However, As important as the measurement of the TLI seems to be, currentmethods of measuring the TLI are impractical and are not physiological.For breast tumors, assays must be conducted within approximately 2 hoursof surgery, precluding a central laboratory from carrying out themeasurement. Generally, the TLI is measured under very high atmosphericpressure in a salt solution to allow penetration of ³ H-thymidine intothe tissue. Under these conditions the tumor loses viability after a fewhours and in many cases it must be assumed that cells capable of cyclingare not measured since the time of measurement is so much less than thegeneration time of the asynchronous cells within the tumor. With regardto other human tumor types, there is very little information regardingmeasurement of proliferation capacity of surgical specimens.

While flow cytometry provides a more rapid method of measuring cellcycle kinetics and cells can also be assessed for aneuploidy, itpresents the following technical problems: (i) Dissociation, eithermechanically or enzymatically, into a single-cell suspension isrequired, resulting in loss of ability to observe tissue architectureand the potential selective loss of one or more specific populations ofcells. Full evaluation of all the heterogeneous cell types of anindividual tumor, including their spatial organization, is of obviousimportance in the development of prognostic tests. (ii) Flow cytometrydoes not unambiguously distinguish between S-phase diploid cells andaneuploid resting or nonviable cells. This becomes an important issue,as studies have demonstrated that tumor cell subpopulations that areenriched in aneuploid cells are largely nonviable by dye-exclusionanalysis. Frankfurt et al., Cytometry, 5: 71-80 (1984); Slocum et al.,Cancer Res., 41: 1428-34 (1981); and Ljung et al., Proc. Am. Assoc.Cancer Res., 28: 34 (1987). (iii) In addition, the S-phase factions ofdiploid tumors are likely to be underestimated by flow cytometry due tocontamination with non-proliferating, nonneoplastic cells. The invasivecapacity of diploid cells in vitro from primary breast carcinomas hasbeen clearly demonstrated. Smith et al., Proc. Natl. Acad. Sci. USA, 82:1805-9 (1985).

The nuclear antigen Ki-67 seems to be present in proliferating breastcancer cells McGurrin et al., Cancer, 59: 1744-50 (1987)!, but itsrelevance to other tissue types is not yet known.

Perhaps most importantly, these techniques measure cells in S phase at asingle point in time (flow cytometry, Ki-67) or after a very shortlabeling time (TLI). Thus, these measurements preclude an estimation ofthe total cell growth fraction of the tumor which may well reflect amore accurate measurement of the proliferative capacity of the tumor.

Importantly, none of the above methods have been applied tosystematically measure the proliferation capacity of normal tissues, inparticular in comparison with adjacent tumor tissues, nor have the abovemethods been applied systematically in comparative measurements ofnormal or tumor tissue proliferation in the presence ofanti-proliferative or other therapeutic agents.

The determination of effective cytotoxic endpoints for chemotherapy isparticularly important when one considers the heterogeneity of celltypes in a tumor tissue and the possible difference betweenproliferating cells and those cells which a not proliferating but areviable in the tumor. Although a cytotoxic agent may exhibiteffectiveness against proliferating cells in an in vitro assay, it mustalso be shown to be effective against non-dividing but viable cells inthe tumor in order to be assured that all cells of the tumor are killed.

Furthermore, for accurate measurements of cytotoxic endpoints foreffective use of cytotoxic agents, the use of prior in vitro assayswhich disaggregrate tumor cells does not provide a reliable testenvironment reflective of the three-dimensional architecture of thetumor tissue. Assays using disaggregated tissue provide equal and readyaccess of the cytotoxic agent to the cells of the tumor, which is nottypical of in vivo tumor tissues.

The use of an in vitro chemosensitivity test system must provide resultsthat can be readily measured and that reliably predict in vivoeffectiveness of the cytotoxic agent being tested.

BRIEF SUMMARY OF THE INVENTION

The present invention provides in improved in vitro assay system todetermine chemosensitivity of a preselected cell population in a normalor tumor tissue to a cytotoxic agent. In particular, the inventionprovides an improved system for measuring chemosensitivity of a tissueto a cytotoxic agent by measuring both the cellular proliferation andcellular viability of the cells from patient tissue biopsies whenassayed in a native histocultured state.

Therefore, a method for measuring the tumor specific effects of an agenton cell proliferation and/or viability is also contemplated. The methodcomprises histoculturing, in separate containers, first and secondportions of tumor tissue samples. The histocultured samples are exposedto one or more concentration of an agent whose effects are beingexamined. The two exposed samples are then treated (contacted) witheither a proliferation marker or a cell viability marker, and the twosamples are then further histocultured for a predetermined period oftime. The percent of cells exhibiting proliferation and the percent ofcells exhibiting viability in the histocultured samples is thendetermined, and, by comparing the results obtained from the treatedtumor cells with results obtained from control untreated tumor cells,the tumor specific effect of the agent on cell viability and/orproliferation is determined.

In preferred embodiments, a normal tissue from a tissue analogous to thestem cells of the tumor is also histocultured to obtain furthercytotoxicity data for the drug being tested.

It has also been found that the determinations as to cellularproliferation and viability made according to the present inventionaccurately predicts the grade, stage and overall aggressiveness of atumor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for measuring the effectivenessof an agent as an chemotherapeutic agent on normal or tumor cells intheir native environment, i.e., as part of a tissue. In carrying outsuch "drug" sensitivity measurements, the effect on the viability andproliferation of cells of the tissue to exposure to one or more givendose levels of one or more given drugs is measured by individuallyculturing aliquots of the same explanted tissue using a plurality ofcontainers, and determining the drug's effect on both proliferation andviability of cells in the tissue. One of the aliquots serves as acontrol and is quantitatively assayed for cell viability and/orproliferation in the absence of drug exposure. Each of the otheraliquots is exposed to the drug at a different dose level and thenquantitatively assayed for viability and/or proliferation by the methodsdescribed herein.

A. Histoculture Methods

Any suitable method may be used for culturing the tissue, so long as thethree dimensional architecture of the tissue to be evaluated ismaintained. Culturing of intact tissue to preserve the three dimensionalarchitecture is referred to herein as histoculture. Maintenance oftissue architecture is important to accurately reflect the in vivogrowth characteristics of the tissue, and in the case of a tumor tissueto reflect the potential in vivo responsiveness of the tumor to a testagent. Typical histoculturing methods include those described by Freemanet al., Proc. Natl. Acad. Sci. USA, 83: 2694-2698, 1986; and Vescio etal., Proc. Natl. Acad. Sci. USA, 84: 5029-5033, 1987 (these references,and others cited herein, are hereby incorporated by reference in thisapplication).

Normal or tumor specimens are typically explanted for histoculture by anaseptic surgical procedure and minced aseptically into about 0.5 to 1.0mm³ pieces. Best results are obtained with approximately cubical 1 mm³pieces. Preferably, multiple portions of a tissue are examined inparallel culture assays because of the heterogeneous nature of mosttissues, particularly tumors. As discussed herein, the method of thisinvention permits the heterogeneity of the tissue to be taken intoaccount.

In order to more effectively produce tumor fragments of suitable sizefor histoculture, a multiblade scalpel can be used consisting of 2 to100 scalpel blades mounted in parallel on the end of a handle, withspaces of 1 mm (for example) between blades. This multi-blade formatallows for more rapid, accurate and reproducible cutting of cubes oftissue 1 mm on a side.

The tissue pieces to be cultured are placed on a suitable supportmaterial in one or more separate containers or wells. The supportmaterial may be any suitable material having a trabecular structure withinterstices capable of delivering aqueous nutrients from a liquid mediumin contact with the support by capillary action to the tissue pieces.The support material may be a suitable mesh formed from a syntheticresin such as nylon, borosilicate glass fiber, polypropylene or anatural organic material such as cellulose or collagen. In addition, thesupport material can be coated with, or covalently linked to,extracellular matrix materials such as fibronectin or laminin. Forexample, fibronectin binds to collagen through strong non-covalentinteractions, and laminin can be linked to gelatinized pig skin usingcrosslinking agents such as dimethyl suberimidate. Preferably, humanfibronectin or human laminin are used for human tissues, allowing a moreaccurate model of the extracellular interactions occurring between atumor and extracellular matrix in human beings.

Preferred materials include gelatinized pig skin, described by U.S. Pat.No. 4,060,081 to Yannas et al., a collagen-containing gel available fromHealth Designs, Inc. (Rochester, N.Y.) or the Upjohn Company (Kalamazoo,Mich.) under the Gelfoam trademark, a homopolysaccharide sponge of thesort described by Leighton, J. Natl. Cancer Inst. 12: 545-561 (1951) andcombinations of collagen-containing gels and homopolysaccharide spongematerials.

Various liquid tissue culture nutrient media capable of supportingtissue cell growth are known in the art. The medium used can either beserum-containing or serum-free with additives such as insulin,transferrin, selenium, estradiol and the like. A culture medium found tobe particularly suitable in the present invention in Eagle's minimalessential medium (MEM) Eagle, Science, 122: 501 (1955) and Eagle,Science, 130: 432 (1959)!.

The tissues are typically histocultured for at least 1-5 days prior toexposing the cells therein to the agent being examined. Culturing istypically performed in a humidified atmosphere at a temperaturecorresponding to that of the body temperature of the animal from whichthe tissue sample came, e.g. 37 degrees C for human tissue samples.Exemplary histoculture conditions are described in the Examples.

B. Cell proliferation Assays

According to the present invention, the histocultured tissue is assessedfor chemosensitivity to a candidate agent by measuring the proliferativecapacity of cells in the tissue upon and/or after exposure to the agent.Proliferation (i.e., multiplication) of the cells occurs by celldivision and therefore may be measured using a variety of proliferationmarkers sensitive to cell division. Furthermore, different types ofcells in the intact tissue may exhibit different degrees ofproliferation upon or after exposure to the agent, providing furtherinformation about the tissue, and in cases of a tumor tissue, about thestage, grade and/or aggressiveness of the tumor.

A typical measure of cell proliferation involves the incorporation of aDNA-synthesis marker into the proliferating cells. That is, the numberof proliferating cells in the tissue is indicated by the number of cellsin the histocultured tissue sample having metabolically incorporated theDNA-synthesis marker into the cellular DNA during cell divisionassociated with cell proliferation.

Proliferation markers for cellular DNA-synthesis are also well known inthe art and include radioactively labeled nucleotides such as ³H-thymidine, ³ H-deoxyadenosine, ³ H-deoxyguanosine, ³ H-deoxycytidine,3-deoxyuridine and the like.

Exemplary conditions for incorporation of DNA-synthesis markers tomeasure cell proliferation, and the analytical methods for assessingproliferation, are described in the Examples.

The assessment of proliferation capacity can be expressed as "growthfraction index" (GFI) of a tissue. In preferred embodiments, the growthfraction index of a tumor tissue correlates to the tumor's in vivo gradeand stage, i.e., the in vivo aggressiveness of the tumor, particularlyin breast and ovarian carcinomas.

The growth fraction index is determined by histoculturing, as describedherein, a sample of cells in an intact tissue. The cells are thentreated with a proliferation marker such as one of the before-describedDNA-synthesis marker, e.g., ³ H-thymidine. The treated cells arecultured for a predetermined period of time and then the percent ofsample cells containing the proliferation marker incorporated into thecellular DNA is determined, that percentage representing the tissue'sintrinsic growth fraction index.

The GFI for a particular tissue is the percent of proliferating cells ina population of total cells in the treated histoculture. GFI isexpressed as a percent and is calculated as the number of proliferatingcells (P) divided by the number of total cells (T) times 100. Theformula for GFI can be expressed as GFI=100×P/T. The GFI value istypically calculated independently for duplicate samples, one a controland the other treated with the agent, after similar culture conditionsand times. A reduction in GFI of 50% or greater for the treated culturescompared to control was indicative of in vitro sensitivity to the drug.

C. Cell Viability Assays

According to the present invention, the histocultured tissue can also beassessed for chemosensitivity to a candidate cytotoxic agent bymeasuring the viability of cells in the tissue upon and/or afterexposure to the agent. Viability of the cells may be measured using avariety of viability markers. Furthermore, different types of cells inthe intact tissue may exhibit different degrees of viability upon orafter exposure to the agent, providing further information about thechemosensitivity of individual cell types within the tissue.

A typical measure of cell viability involves detecting cellularmetabolism in the cell, such as by using metabolic markers indicative ofviability. Such markers of cell viability are well known and includeassays of cell membrane integrity (viable dye exclusion orincorporation), protein synthesis (uptake of labeled amino acids intonewly synthesized protein), and other metabolic markers. The number ofviable cells in each tissue sample is indicated by the number of cellsthat incorporate a metabolic marker or that otherwise exhibit viability.

i. Protein Synthesis Markers

In one embodiment, the incorporation of a label into a cellular proteinduring protein synthesis (i.e., a protein-synthesis marker) isindicative of cell viability insofar as cellular metabolic activity isoccurring upon incorporation of the marker. Metabolic markers forcellular protein synthesis are well known in the art and includeradioactively labeled amino acids such as ³⁵ S-methionine, ¹⁴ C-alanine,¹⁴ C-glycine, ¹⁴ C-glutamic acid, ¹⁴ C-proline, ³ H-leucine, ³ H-serineand the like. Exemplary protein synthesis markers are used as describedin the Examples for measuring viable cells in a histocultured tumortissue sample.

ii. Viability-Specific Dyes

In another embodiment, dye exclusion or dye incorporation can beutilized to indicate the presence of viable cells in a tissue sample.For example, cells which incorporate a dye specific for dead cells arescored non-viable, and cells which exclude the dead cell-specific dyeare scored as viable. Conversely, dyes specific for viable cells can beused to directly on the histoculture to indicate the presence of viablecells.

As used herein, the phrase "specific for dead cells" means that theindicator is taken up or incorporated only into dead, non-viable cells.

Typically, dyes specific for dead cells are compounds with a high ioniccharge and low permeability such that the dyes cannot permeate intactcellular membranes. When cells die, the membrane is structurally orfunctionally ruptured such that dyes specific for dead cells gain accessto the intracellular space where they bind to intracellular componentssuch as nuclear membranes.

A preferred dead cell-specific indicator is a dye capable of opticaldetection. See, e.g., Handbook of Fluorescent Probes and ResearchChemicals, ed. by R. P. Haugland, Molecular Probes, publisher, Eugene,Oreg. (1989-1991). A preferred dead cell-specific dye is a fluorescentdye such as propidium iodide (PI), ethidium bromide (EtBr), ethidiumhomodimer (5,5'-diazadecamethylene) bis(3,8-diamino-6-phenyl-phenanthridium) dichloride, dihydrochloride! andthe like. Most preferred is propidium iodide for use as a dye specificfor dead cells. Propidium iodide and other dyes specific for dead cellsare well known in the art and are commercially available (MolecularProbes, Eugene, Oreg.).

iii. Metabolism Markers

In another embodiment, cell viability can be measured by detecting theconsumption of a metabolite from the histoculture medium. A typicalconsumption endpoint metabolite is glucose.

In a related embodiment, cell viability can be measured by the use of ametabolic substrate which is converted into a detectable product uponreaction with a cellular component indicative of cell viability. Anexemplary metabolic substrate is any of the tetrazolium salts which areconverted by the action of cellular dehydrogenases into a detectableformazan compound.

A suitable tetrazolium salt solution is prepared by dissolving theselected salt in a suitable stock solution such as saline, which ispreferably phosphate-buffered. Typical tetrazolium salts include 3-4,5-dimethylthiazol-2-yl!-2,5-diphenyltetrazolium bromide (MTT),2,2',5,5'-tetra-p-nitrophenyl-3,3'-3,3'-dimethoxy-4,4'-diphenylene!ditetrazolium chloride (TNBT), 3,3'-3,3'-dimethoxy(1,1'-bi-phenyl)-4,4'-diyl!-bis2,5-diphenyl-2H-tetrazolium! dichloride (tetrazolium blue; TB),2,3,5-triphenyltetrazolium chloride (tetrazolium red; TR),2,5-tiphenyl-3- α-naphthyl!-tetrazolium chloride (tetrazolium violet;TV), 2- 4-iodophenyl!-3- 4-nitrophenyl!-5-phenyltetrazolium chloride(INT), 2,2',5,5'-tetraphenyl-3,3'- p-diphenylene!ditetrazolium chloride,2,2'-di-p-nitrophenyl-5,5'-diphenyl-3,3'-3,3'-dimethoxy-4,4'-diphenylene!ditretrazolium chloride (NBT), 2,2'-dip-nitro-phenyl!-5,5'-di p-thiocarbamylphenyl!-3,3'-3,3'-dimethoxy-4,4'-biphenylene!ditretrazolium chloride (TC-NBT), andmixtures thereof.

The selected tetrazolium salt, preferably MTT, is dissolved in asuitable medium, typically phosphate-buffered saline (PBS). Thepreferred concentration is from about 4 mg/ml to 12 mg/ml, with optimumresults at about 8 mg/ml. The solution is preferably filtered beforeuse. From about 50 to 150 μl of the MTT solution is added to each tissuesample. Best results are obtained with about 100 μl. The samples arepreferably again incubated for about 1 to 3 hours under incubationconditions as described herein.

The samples are then removed, frozen, and frozen sections of about 4μare made in a conventional manner. The resulting slides are then dippedin a suitable fluorescent dye solution for from about 30 seconds to 2minutes to allow the stain to penetrate the section and stain all thecells in the section, providing a detectable indication of the totalnumber of cells in the section, dead or alive. A 1 minute dip ispreferred. Typical suitable stains for labelling cells are any stainthat is specific for cells and which is detectable by light microscopy.Preferred are the family of fluorescent dyes that intercalate DNA andthereby fluoresce, indicating the presence of nuclei. Such dyes aregenerally known as high-affinity nucleic acid stains. Exemplaryfluorescent dyes are ethidium bromide (EtBr), ethidium homodimer,propidium iodide (PI), and the like dyes available from MolecularProbes, Inc., (Eugene, Oreg.). Particularly preferred is PI, and isutilized as exemplary herein. In each case where a different stain isused, a filter system is selected to produce light of wavelengths towhich the fluorescent dye is sensitive.

The slides are then dried and examined with a microscope, typically atabout 200 X utilizing a video camera attached to the microscope. Thesections are first illuminated with polarized light, typically generatedby a mercury lamp. The cells containing formazan crystals due tometabolically reduced tetrazolium salt brightly reflect polarized light.The images are analyzed by pixel image analysis, typically as describedby Hoffman et al, Proc. Natl. Acad. Sci. USA, 86: 2013-2017, (1989).

The slides are also analyzed for fluorescence using light passed througha filter providing the proper wavelength to activate the selectedfluorescent dye. The number of bright pixels from the fluorescence orreflectance measurement is calculated using a modified Fas-Com versionof the P-See program from The Microworks, Del Mar, Calif., typically runon an IBM PC XT type computer. Cell morphology can be observed due tothe fluorescence whereby cancer cells can be distinguished from stromalcells. The slide images are digitized by a conventional digitizer boardand the area of brightness corresponding to the number of labeled orbright cells is calculated as the area of enhanced pixels by the Fas-Comprogram. The area of enhanced pixels is proportional to the number oflabeled cells and may represent the number of viable cells when labeledwith a viability marker such as MTT, and represents the total number ofcells when labeled with a total cell specific dye. The ratio of formazanbright pixels to fluorescent dye bright pixels can be calculated foreach drug tested and compared to a control value to obtain the amount ofdrug-induced inhibition.

For example, the sensitivity of a tumor tissue to a cytotoxic drugexpressed as viable cell inhibition rate can be determined by using theformula: ##EQU1## where PIAFC is pixel image analysis of formazancrystal reflection and PIAFD is pixel image analysis of fluorescent dyeemissions.

Using a viability marker as described above, one can also determine theviable cell index (VCI) of the cells in the tissue following exposure toa chemotherapeutic agent of interest, when compared to untreated controltissue culture. VCI is a measure of the percent of viable cells in apopulation of total cells in the treated histoculture. VCI is expressedas a percent and is calculated as the number of viable cells (V) dividedby the number of total cells (T) times 100, where T is viable cells (V)plus dead cells (D). The formula for VCI can be expressed asVCI=100×V/(V+D). The VCI value can be calculated for duplicate samples,one a control and the other treated with the agent, after similarculture conditions and times. A reduction in VCI of 50% or greater forthe treated cultures compared to control was indicative of in vitrosensitivity to the drug.

Typically, the number of dead cells (D) is measured by counting cellslabeled with a dye specific for dead cells, as described herein.Similarly, the number of viable cells is measured by counting cellslabeled with a dye or metabolic marker specific for viable cells. Asused herein, the phrase "specific for viable cells" means that theindicator/marker is taken up (incorporated) or detectable in living, butnot dead, cells.

The indicator specific for viable cells may be a metabolic precursor ora non-metabolite that gains access to living cells.

A preferred non-metabolite indicator specific for viable cells is a dyethat is capable of optical detection. Any dye recognized in the art asbeing specific for viable cells can be used in accordance with thetoxicity assay of this invention. See, e.g., Handbook of FluorescentProbes and Research Chemicals, ed. by R. P. Haugland, Molecular Probes,publisher, Eugene, Oreg. (1989-1991).

In a preferred embodiment, the dye is a fluorescent dye. Exemplaryviable cell-specific fluorescent dyes are BCECF-AM B-1150:2'7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethylester!, calcein-AM (C-1430:glycine-N,N'-((3',6'-dihydroxy-3-oxospiro(isobenzofuran-1(3H),9'-(9H)xanthene)-2,7-diyl)bis(methylene))bis(N-carboxymethyl)-acetoxymethylester), CFDA (C-195: 5-carboxyfluorescein diacetate) acridine orange(A-1301: 3,6-acridinediamine, N,N,N',N'-tetramethyl-monohydrochloride),calcein blue (H-1426:7-hydroxy-4-methylcoumarin-8-methyleneiminodiacetic acid), Fura-2AMF-1201: 5-oxazolecarboxylic acid2-(6-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-(2-(2(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-methylphenoxy)ethoxy)-2-benxofuranyl)-,(acetyloxy)methylester, fluorescein diacetate (F-1303) or carboxy calcein blue AM(C-1431: 7-hydroxy-4-carboxymethylcoumarin-8-methyleneiminodiacetic acidacetoxymethyl ester) and the like. Such dyes are well known in the artand are commercially available (Molecular Probes, Eugene Oreg.).Particularly preferred are the dyes BCECF-AM or calcein-AM. The numeralsin the parenthesis indicates the product number for the listedfluorescent dyes that are available from Molecular Probes.

In one embodiment, the incorporation or uptake of fluorescent dyesspecific for viable cells depends upon metabolic activity of the viablecell. In accordance with this embodiment, non-fluorescing dyes are takenup by viable cells and converted to a fluorescing product by anintracellular enzyme such as an esterase. The presence of intracellularfluorescence thereby indicates viability.

The various types of tissues to which the present invention isapplicable include normal tissues as well as primary or metastatictumors, including solid tumors (both carcinomas and sarcomas) and thelike.

The various types of carcinomas (adeno, squamous and undifferentiatedvariants for carcinomas of various sites), to which the present assayare applicable include, for example, adrenal, bladder, breast, colon,kidney, lung, ovary, pancreas, prostate, thyroid, upper airways (headand neck), uterus (corpus and cervix), bile ducts, choriocarcinoma,esophagus, liver, parathyroid, rectum, salivary glands, small bowel,stomach, testis, tongue and urethra. The various types of sarcomas andother neoplasms to which the present assay are applicable include, forexample, diffuse lymphomas, Ewing's tumor, Hodgkin's disease, melanoma(melanotic and amelanotic), multiple myeloma, nephroblastoma (Wilm'stumor), neuroblastomas, nodular lymphomas, rhabdomyosarcoma,angiosarcoma, brain tumors (gliomas), chondrosarcoma, dysgerminoma,fibrosarcoma, leiomyosarcoma, liposarcoma, medulloblastoma,mesothelioma, osteosarcoma, retinoblastoma and thymoma.

Typically, the tissue whose cell viability and/or proliferationcharacteristics are to be determined is explanted by an aseptic Surgicalprocedure and a portion thereof is divided into sections having a volumeof about 0.5 to about 10, preferably a volume of about 1.0 to about 8.0,more preferably 1.0 to 2.0 cubic millimeters.

When tumors are being assayed, it is important to examine multipleportions of the tumor in separate assays because tumors are veryheterogeneous.

After cubing, the explanted tissue is divided into aliquots (portions),typically at least about six, one of which is typically designated acontrol that receives no exposure or contact with the compound beingexamined. The aliquots are then histocultured on hydratedextracellular-matrix-containing gel so that the three-dimensionalintegrity of the tissue is maintained.

Drug exposure of the cells for the purposes of the viability and/orproliferation measurements is preferably carried out prior to treatingthe tissue samples with the viability and proliferation markers. Theprocedure involves incubating the tissue sample with a predeterminedamount (determinate concentration) of the drug for a predeterminedperiod of time (determinate time period) and thereafter separating thetissue sample from the drug, and preferably washing the tissue samplefree of residual drug.

The phrase "drug exposure dose level", as used herein, refers to thequantitative product of the drug concentration (e.g. in μl) and the timeof the exposure period (e.g., in hours or minutes). The drugconcentrations and exposure times are typically calculated frompharmacological data to the simulate in vitro the drug exposure doselevel achieved in vivo. Typically, it has been found that the drugexposure dose level required in carrying out the drug sensitivitymeasurements in accordance with the assay of the present invention, isat a maximum of only 5 to 10% of the clinically achievable drug exposuredose level for the known anticancer drugs which have been tested in thepresent system.

The drug sensitivity measurements as described above can be carried outin a manner which enables the determination, for any given drug, of a"drug sensitivity index", which is indicative of the antineoplasticactivity of the given drug against the specific human tumor from whichthe explanted cells were obtained. This procedure involves carrying outthe drug sensitivity measurements of a plurality of dose levelsextending over a multi-log range, and then using the results of thesemeasurements to plot a curve of percent survival (the percentage of theassay count resulting from drug exposure versus the assay count of thecontrol in the absence of drug exposure) versus drug exposure doselevel. The "drug sensitivity index" of the given drug is thenquantitated by measuring the area under such curve out to a definedupper limit which is correlated to the clinically achievable peak drugexposure dose level for that drug.

The sensitivity index obtained in the above-described manner is highlyindicative of the antineoplastic activity of the drug against thespecific human tumor from which the explanted cells were obtained, witha low sensitivity index indicating high antineoplastic activity.

After histoculturing the cells in the presence of the agent beingexamined (about 1 to 4 days) the samples are treated (cultured in thepresence of) the proliferation and/or viability marker.

After the cells of the tissue samples have been exposed to the drug(s),treated with the proliferation and viability markers and subsequentlyhistocultured, the samples are typically fixed in the tissue fixativesuch as formalin, embedding in paraffin or the like and sectioned on amicrotine. The sections are then assayed for the percentage of cellspositive for the presence of the viability of proliferation marker andfor calculating VCI and GFI as described herein.

When the viability and/or proliferation markers contain radioactivelabels, the sections are then treated with nuclear-track emulsion suchas NTB (Kodak), and developed. When radioactive labels and nuclear-trackemulsions are used, this can be accomplished by light-polarizedmicroscopy.

Where the markers are fluorescent dyes, the sections are stained withthe appropriate dye(s), analyzed under fluorescence microscopy, and thenumbers of dead and/or live cells are counted as described herein.

In preferred embodiments, the magnified image can be digitized byprocessing it through a video camera operatively linked to a computercapable of digitizing the image for analysis.

Preliminary evidence indicates that the in vitro assay system of thepresent invention has utility for the in vitro prediction of clinicalresponse to cancer chemotherapy, as well as the screening of newanticancer drugs for clinical trial. For example, in treating a specificpatient for a specific tumor, the explanted cells obtained from a biopsyof such specific tumor can be assayed in accordance with the presenttechnique, whereby drug sensitivity measurements are made according tothe present invention for a plurality of different anticancer drugswhich are potentially clinically effective for the chemotherapeutictreatment of the specific tumor. After determining the relative drugsensitivity indices (e.g., GFI and/or VCI) for each of the various drugstested, these sensitivity indices may be used for predictably selectingthe most promising and/or effective of the drugs to be used for thechemotherapeutic treatment. In preliminary clinical trials of thistechnique, both retrospective and prospective, the correlation foundbetween the in vitro prediction and the in vivo response for aparticular agent by a particular tumor tissue type was improveddramatically where viability and proliferation were both evaluated.

In another embodiment, the present invention contemplates a kit for thein vitro determination of viability and/or proliferation of cells asdescribed herein. The kit contains, in an amount sufficient for at leastone assay, an agent to be tested for its effects on viability and/orproliferation, a viability and/or proliferation marker, and a containerin which to perform the assay.

EXAMPLES

The following examples are given for illustrative purposes only and arenot intended to be limiting unless otherwise specified.

1. Cell Proliferation in Histoculture

Various normal and tumor tissue specimens were ex-planted from humanpatients as described by Freeman et al., Proc. Natl. Acad. Sci. USA, 83:2694-98 (1986); and by Vescio et al., Proc. Natl. Acad. Sci USA, 84:5029-33 (1987). (The teachings of all of the references cited herein arehereby incorporated by reference). Briefly, after tissues weresurgically removed, they were divided into 1- to 2-mm-diameter piecesand placed on top of previously hydrated extracellular-matrix-containingflexible gels derived from pigskin (Gelfoam, Upjohn) to form athree-dimensional culture. Eagle's minimal essential medium (MEM)containing Earle's salts, glutamine, 10% fetal calf serum, nonessentialamino acids, and the antibiotics garamycin and claforan was added to thecultures such that the upper part of the gel was not covered, andcultures were maintained at 37 degrees C in a carbon dioxide incubator(95% sterile air/5% CO₂) to allow the explanted tissue specimens togrow.

Cells within the three-dimensional cultures capable of proliferationwere labeled by administration of a combination of ³ H-thymidine and ³H-deoxyuridine (2 μCi each; 1 Ci=37 GBq) (Vescio et al., Proc. Natl.Acad. Sci. USA, 84: 5029-33, 1987) for 4 days after 10-12 days inculture. Cellular DNA is labeled in any cells undergoing replicationwithin the tissues. After 4 days of labeling, the cultures were washedwith phosphate-buffered saline, placed in histology capsules, and fixedin 10% Formalin. The cultures were then dehydrated, embedded inparaffin, and sectioned by standard methods, and the sections wereplaced on slides.

The sections on the slides were then deparaffinized and prepared forautoradiography by coating with Kodak NTB-2 emulsion in the dark andexposed for 5 days, after which they were developed. After developingand rinsing, the sections were stained with hematoxylin and eosin.

The sections were then analyzed by determining the percentage of cellsundergoing DNA synthesis in the various treated versus non-treated tumortissue cultures, using a Nikon or Olympus photomicroscope fitted with anepi-illumination polarization lighting system. Replicating cells wereidentified by the presence of silver grains, visualized as bright greenin the epi-illumination polarization system, over their nuclei due toexposure of the NTB-2 emulsion to radioactive DNA and subsequent greenreflection of the polarized light by the silver grains. The aboveprocedures produce a histological autoradiogram showing cellularproliferation of specific cells in the histocultured tumor explanttissue.

The large majority of tumors cultured in the native-state systemdemonstrate at least some areas of high cellular proliferation and areintratumorally heterogeneous with regard to proliferation capability.Tumors tested include tumors of the colon, ovaries, pancreas, bladder,kidney, brain, and parotid, and also include small-cell lung carcinomaand Ewing sarcoma. In all cases, three-dimensional tissue organizationrepresentative of the original tissue is maintained throughout theculture period. A high degree of detection of radiolabeled proliferatingcells is afforded by the epi-illuminescence polarization microscopy,which enhances detection of the autoradiographic exposed silver grainsby the scatter of incident polarized light.

The proliferation capacity of a metastatic colorectal tumor exhibitedhigh labeling in culture. More than 90% of the cells in the observedculture preparation had proliferated during the labeling period of thisrelatively undifferentiated colon metastasis to the liver.

The proliferation capacity of a small-cell lung tumor exhibited themaintenance of the two major classes of oat cell types: the classicsmall cells and the more elongate fusiform cell types, each having ahigh degree of cell proliferation.

The proliferation capacity in ovarian carcinoma consistently exhibitedan extremely high index of proliferation of the epithelial cells whilethe stromal cells remained quiescent. The histological autoradiogramshowed the high proliferative capacity of the ovarian carcinoma cellswhich have invaded the supporting gel matrix. This invasive behavior maymimic the way ovarian tumors frequently invade the peritoneal wall invivo.

The proliferation capacity in miscellaneous tumors, including those ofthe pancreas, bladder, kidney, brain, and parotid gland, and a Ewingsarcoma exhibited the intricate gland formations containingproliferating cells in many of these cultured tumors.

It is important to note that distinctions can be observed in theprepared autoradiograms between proliferating malignant cells and normalcell types, such as for the breast tumor epithelial cells and normalstromal cells.

An additional important observation in these studies is that normaltissues culture and proliferate well. Explanted tumor and adjacentnormal tissue from the breast of patient 431 were compared. Extensivecell proliferations were noted to be present in the normal tissues.However, a higher level of tissue organization was observed to bemaintained in the normal tissues. With this system if is now possible tocompare tumor and normal biology--for example, nutritional requirements,growth factor requirements, and metabolic pathways. Also of criticalimportance, it is now possible to compare the antitumor selectivity ofpotential neoplastic agents by comparing tumor and normal response todrugs, using cell proliferation as an endpoint as described in Example3.

These results have demonstrated a generalized system for measurement ofproliferation capacity for all the major types of human tissues inrelatively long-term culture. As mentioned above, all cultures describedin this report have been viable in culture for 14 days, which is arelatively long period. Greater periods of culture can be achieved withmost tissue specimens (data not shown). Greater than 90% of surgicalspecimens can be cultured and analyzed for proliferative capacity withthis system.

This native-state culture system, with the aid of polarizationmicroscopy, allows a high probability of detecting potentialproliferative cells.

For image analysis of proliferating cells, a video camera was attachedto the microscope. Autoradiograms prepared as above using breastcarcinoma tissue were then viewed under polarizing light withoutbright-field light, thereby visualizing only the radioactive cells whichhave exposed silver grains of the nuclear-track emulsion. Theradioactive cells brightly reflect the polarized light. The resultingimage was analyzed by a computer-assisted image analysis apparatus inwhich the image was first digitized by a digitizer board, and then thearea of brightness corresponding to the number of labeled or brightcells was calculated as the area of enhanced pixels by the Fas-Comversion of the P-See program (The Microworks, Del Mar, Calif.) run on anIBM PC XT compatible computer. The area of enhanced pixels isproportional to the number of labeled cells.

With the image analysis system the autoradiograms were automaticallyanalyzed for the number of labeled proliferating cells. With thebright-field and polarized light microscopy, the labeled cells of acultured breast tumor appear bright green. With epi-illuminationpolarization microscopy using polarized light without bright-field, onlythe labeled cells were visualized. The image of the labeled cells wasthen digitized through a video camera and the P-See program. The area ofbrightness or enhanced pixels was then automatically determined by theFas-Com program. The area of enhanced pixels is proportional to thenumber of labeled cells, enabling the automatic counting of labeled,proliferating cells.

An important aspect of the culture system is the use of a flexibleextracellular-matrix-containing gel on which to ex-plant the tumors.Other investigators have noted that flexibleextracellular-matrix-containing substrata are critical to growth andfunction of differentiated cells. Li et al., Proc. Natl. Acad. Sci. USA,84: 136-40 (1987); Davis et al., Science, 236: 1106-9 (197); Schaefer etal., Cancer Res., 43: 279-86 (1983); Schaefer et al., Differentiation,25: 185-92 (1983); and Leighton, J., in Tissue Culture Methods andApplications, eds., Kruse et al., (Academic, New York), pp. 367-71(1973).

The general principles here are applicable to all types of humantissues, allowing the accumulation of potential important biological andclinically prognostic information. In addition, it should be noted thatmany of these tumors have high capabilities of cell proliferation. Theeventual understanding of the deregulation permissive for suchproliferation should be facilitated with the system described here andallow us a deeper understanding of the changes occurring in oncogenesis.

2. Determining Cell Viability and Proliferative Capacity in Native-StateTissue Culture

Tumor tissue specimens from a patient having breast carcinoma wereobtained as described in Example 1, divided into 1 mm diameter pieces,and were each placed onto a flexible gel matrix to form a threedimensional culture. Duplicate cultures are prepared of each specimen,and 8 microcuries (μCi) of either ³⁵ S-methionine or ³ H-thymidine wasadded to each culture that includes 2 milliliters (ml) of culture mediumcontaining the added radiolabel. The labeled cultures were maintained asbefore for four days, the excess radiolabel was then rinsed off of eachcultured specimen using a series of phosphate-buffered saline (PBS)rinses and the cultures were each processed for histological andautoradiographic visualization as described in Example 1.

The prepared cultures were then analyzed by using the computer programFas-Com for analysis after digitizing as described in Example 1. Themeasure of ³⁵ -methionine incorporated into cultured cells allows thedetermination of cellular protein synthesis and is therefore a measureof cell viability. The measure of ³ H-thymidine incorporation intocultured cells allows the determination of DNA synthesis and istherefore a measure of cell proliferation.

Tissue specimens cultured in the presence of ³⁵ S-methionine or ³H-thymidine incorporated radiolabel in the portions of the tissuecontaining viable or proliferating cells, respectively, or both.Non-viable or non-proliferating cells did not incorporate theirrespective labels and did not present silver grains on visual inspectionof prepared specimens, nor present proliferating cells as bright greenobjects when analyzed in the epi-illumination polarization system.

The percentage of cells labelled with protein synthesis marker out ofthe total number of cells was determined in each culture. Similarly,percentage of cells labelled with the DNA-synthesis marker out of thetotal number of cells was determined in each culture.

The extent of proliferation measured by the in vitro native-stateculturing system correlates with the grade and stage of the tumor: themore malignant the tumor, the higher the proliferation measured invitro. The extent of proliferation can be expressed as a growth fractionindex (GFI), measured as the percentage of proliferating cells presentin a population of the total number of cells present in a selected fieldviewed by the microscope. Therefore measured GFI can be used to predictthe clinical progression of the human cancer tissue tested. The Fas-Comprogram analysis provides a quantitative means that is semi-automated todetermine the proliferative capacity or viability of a tumor, and isideally suited to provide GFI data.

Proliferation and viability analyses were conducted on numerousexplanted breast tumor tissues graded as metastatic or primary, and alsograded as poorly or moderately differentiated to generate an average GFIfor each type of tumor tested. Whereas metastatic tumors averaged0.437±0.149 GFI, primary tumors exhibited a lower average of 0.282±0.138GFI. Similarly, whereas poorly differentiated tumors averaged0.372±0.150 GFI, moderately differentiated tumors averaged 0.220±0.094GFI. The results indicate that GFI correlates with tumor severity andclinical prognosis.

3. Determining Drug Response Using The Native-State System For MeasuringViability And Proliferative Capacity Of Tissues In Vitro

Tissue specimen cultures were established using various tumor tissues asdescribed in Example 2. After the fourth day of culturing, multiplecultures of each tissue were further cultured in the presence of a drugas indicated below at the indicated concentrations for an exposure timeof 24 hours.

Cultures were then washed in culture medium to remove excess drug,cultured in the absence of drug for 3 days to allow the cells to recoverfrom transient drug effects, and were then labeled as described inExample 2. After labeling, the cultures were processed as before inExamples 1 and 2 to visualize the degree of viability and proliferativecapacity in the specimens when cultured in the presence of the drug.

The cells cultured from the ovarian carcinoma tissue of a patient havingovarian carcinoma were tested by the above methods for cellproliferation in the presence of cisplatin at 1.5 μg/ml, 5-fluorouracilat 4 μg/ml, melphalan at 10 μg/ml, methotrexate at 22.5 μg/ml orthiotepa at 60 μg/ml. The results showed a decrease in the detectablesignal generated by both ³ H-thymidine and ³⁵ S-methionineincorporation, indicating an inhibition of both proliferation andviability. A diminution in cell proliferation is expressed as a decreasein the GFI, when compared in the GFI for the same specimen cultured inthe absence of the drug. A diminution of cell proliferation was observedin an amount of 90% using cisplatin, 99% using 5-fluorouracil, 70% usingmelphalan, 70% using methotrexate and 90% using thiotepa. Patient S.D.having ovarian carcinoma produced explanted tumor tissue that wasinhibited by more than 70% in cell proliferation by melphalan at 10μg/ml, responded to therapy using melphalan, and exhibited a decrease intumor size during treatments. Therefore a clinical correlation wasdemonstrated between in vivo responsiveness of the tumor to the drug andthe in vitro native-state drug responsiveness.

The cells cultured from the tissue of a patient having breast carcinomawere tested as above in the presence of drug, and resulted in thefollowing diminutions in cell proliferation shown in the parenthesis:Adriamycin at 290 ng/ml (90%), 5-fluorouracil at 4 μg/ml (90%),melphalan at 1 μg/ml (80%), methotrexate at 2.25 μg/ml (70%) orvincristine at 23 μg/ml (70%).

Patient D.H. was diagnosed as having breast carcinoma and was determinedto be non-responsive to in vivo therapy with either 5-fluorouracil oradriamycin. Cellular proliferation of patient D.H.'s breast carcinomatissue was not inhibited significantly (i.e. greater than 90%diminution) by culturing as above in the presence of either5-fluorouracil or adriamycin. Therefore, there was a clinicalcorrelation between in vivo responsiveness and in vitro diminutions ofcell proliferation.

The cells cultured from the cancerous tissue from a patient (V.S.)colon/rectal cancer were tested as above in the presence of the variousindicated drugs, and resulted in the following diminutions in cellproliferation shown in the parenthesis: 5-fluorouracil at 4 μg/ml (90%),mitomycin C at 1 μg/ml (90%), and BCNU at 2 μg/ml (90%). Patient V.S.was diagnosed as having colon carcinoma and was determined to benon-responsive to in vivo therapy using 5-fluorouracil. Cellularproliferation of V.S.'s colon carcinoma cells was not inhibitedsignificantly, i.e., greater than 90%, by culturing the explants asabove in the presence of 5-fluorouracil. A clinical correlation wasagain observed between in vivo and in vitro responsiveness.

The above results show that cellular proliferation can be used as ameasure of a tumor's drug responsiveness, where an active drug inhibitscellular proliferation. Where cells are not in a proliferative state,the viability of the tumor tissue as measured by ³⁵ S-methionineincorporation can be used to indicate the tumor's drug responsiveness.The extent of viability can also be used to determine an endpoint formaximum responsiveness.

4. Fluorescent-Dye Endpoint Assay to Determine Chemosensitivity by CellViability

A viability cell index (VCI) was also determined for histocultured tumortissue by using fluorescent dyes to indicate the endpoint in achemosensitivity assay. A first dye that is viable cell-specific and asecond dye that is dead cell-specific are used to determine the VCI forthe tumor tissue in the presence of a chemotherapeutic agent.

The viable cell-specific dye BCECF-AM,2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, is activated tofluorescence by non-specific esterases present only in living cells. Thedead cell-specific dye PI, propidium iodide, enters only non-viablecells whose plasma membranes are leaky. Both dyes were added to thehistoculture at 15 uM, and the cultures were analyzed by fluorescenceconfocal microscopy after 20 minutes of dye staining. Both dyes are usedsimultaneously on the same sample because the emission spectra ofBCECF-AM and PI are different. The histocultured samples were observedusing a MRC-600 Confocal Imaging System (Bio-Rad) mounted on a NikonOptiphot fluorescent microscope fitted with a 10X PlanApo objective anda Nikon DN-510 B-2A fluorescence filter.

The fluorescence endpoint was determined using various drugs in adose-response on histocultured human tumors for 24 hours. Samples wereprepared for histoculture as described in Example 1. Thereafter, theviable cell index (VCI) was determined for both treated and control(untreated) histocultured samples.

5. ³ H-Thymidine Endpoint Assay to Determine Chemosensitivity by CellProliferation

Using the DNA-synthesis marker procedure described in Example 1,proliferation was measured in a series of human tumor histoculturesfollowing exposure to a variety of drugs as described in Example 4 andthe proliferation index was calculated as a growth fraction index (GFI).

The samples were analyzed with confocal microscopy as described inExample 1 to count the number of proliferating cells in thehistoculture. For each drug concentration tested, 1 to 3 microscopefields were observed and counted that contained the highest number oflabeled cells in order to identify the areas in the heterogeneous tumorcultures having the least drug sensitivity. Control (untreated) cultureswere evaluated and counted in the same manner. Six replicate cultureswere evaluated for each drug concentration to determine a statisticallysignificant in vitro drug response. An endpoint for in vitro drugresponsiveness was the concentration at which a 50% reduction in GFI wasobserved when compared to control samples.

In a representative study, the GFI endpoint correlated well with aclinical study using cisplatin to treat patients having head and neckcancers. Patients with head or neck cancer were biopsied to produce ahistoculture sample that was tested in the GFI endpoint assay asdescribed in Example 1. Twenty-three patients who were treated withcisplatin were studied using the in vitro GFI assay, and ten out oftwelve patients who tested as sensitive by GFI responded clinically(83%). In addition, seven out of eleven patients who tested as resistantby GFI demonstrated no clinical response (64%).

6. In Vivo Drug Response Assay

In order to assess the effectiveness of the present methods, in vivodrug dose-responsiveness was conducted and compared to the in vitroindexes generated by the present methods. To that end, the human tumorsfragments of about 27 mm³ described herein were inoculated under etheranesthesia by means of a trocar needle into the subcutaneous tissue onthe back of a nude mouse.

All drugs were dissolved in 0.2 ml of physiological saline andadministered at a schedule of q4dx3 i.p. except for doxorubicin, whichwas given i.v. Dosages administered were 3 mg/kg for mitomycin C, 4mg/kg for doxorubicin, 50 mg/kg for 5-FU, and 80 mg/kg forcyclophosphamide, which were determined to be the maximum tolerabledoses for nude mice when injected on the indicated schedule. Melphalanwas used as the in vitro surrogate for cyclophosphamide which requiresin vivo metabolic activation and cannot be tested readily in vitro.

After inoculation of the tumor tissue, and during administration of thedrug, tumors were measured in the animal (length and width) with asliding caliper three times weekly by the formula: tumor weight (W) inmg equals length (L) in mm times width (W)!² divided by 2, or W=L×W² /2.Typically, when tumors reached 100-300 mg, about 2-3 weeks afterinoculation, tumor-bearing mice were randomized into test groups of sixmice each. The relative mean tumor weight (RW) was calculated as Wi/Wo,where Wi is the mean tumor weight of a group at any given time (i) andWo is the mean tumor weight at the initial treatment. Antitumor effectof the drug was expressed as a ratio of test to control RW, or Trw/Crw,when the ratio was at its lowest value during the treatment using Crwvalues obtained at the same time period as Trw. Antitumor activity wasconsidered positive when the lowest Trw/Crw during the experiment wasless than 42% of control reflecting a 25% reduction of the diameter ofthe tumor.

7. Comparison of Chemosensitivity Indexes

The chemosensitivity indexes GFI, determined as described in Example 5,and VCI, determined as described in Example 4, were obtained for avariety of histocultured human tumor tissue samples that were alsotested for drug responsiveness in vivo as described in Example 6.

Using human small cell lung cancer (Lu24), it was determined thatwhereas doxorubicin produced a dose-response curve when assayed by bothVCI or GFI, 5-FU exhibited relative resistance using VCI, even at highdosages of about 40 ug/ml (10X), where 1 X indicates the level of drugachievable in blood. Thus, different drugs differ in effectivenessagainst a particular tumor tissue when assayed in vitro.

Using human colon carcinoma (1863) it was determined that whereas 5-FUproduces a dose-response of sensitivity by the tumor to 5-FU whenassayed using the GFI assay, the tumor is not significantly responsiveto 5-FU in the VCI assay. Thus, the GFI endpoint can lead to a falsepositive result when considered alone without the VCI endpoint data.

A panel of seven human tumors were similarly tested, including gastriccancers St-4 and St-40, colon cancer Co-4, breast cancer MX-1, and lungcancers Lu130, Lu24 and H69, which were all established as xenograftlines. These human tumors were evaluated for responsiveness to mitomycinC (MitC), doxorubicin (Dox), 5-fluorouracil (5-FU), cisplatin (Cis), andmelphalan (Mel) using the in vivo assay, and the in vitro assaysdetermining GFI and VCI. Overall, there was a reasonable degree ofcorrelation between in vivo sensitivity and the drug responsivenessaccording to either VCI or GFI assays. For example, averaged over alltumor types (7 types) and all drugs (5 drugs), VCI was accurate 73% ofthe time and GFI was accurate 63% of the time, when compared to the invivo result.

Notably, however, depending upon the tumor and the drug tested, a VCIend point and a GFI end point were occasionally observed to giveopposite results regarding drug sensitivity. For example, in some casesVCI gave a false positive result whereas GFI gave a true negative result(i.e., the tissue was resistant to the drug when administered in vivo).Other combinations of opposing drug responsiveness results were alsoobserved. For example, resistance with VCI and sensitivity with GFI todrug responsiveness was observed in 39.3% of the cases studied, whereasthe reverse of an indication of sensitivity with VCI and resistance withGFI was observed in only 8.4% of the cases.

Based on the data, it is seen that the combination of both GFI and VCIsubstantially increases the reliability of the in vitro assay forpredicting in vivo effectiveness of a particular drug against aparticular tumor type by reducing the incidence of false positiveresults (i.e., incorrect indication of drug sensitivity). For example,it was observed that 55% of the false readings produced using a VCIassay were identified as results contradictory with in vivo data by theGFI endpoint assay, and 75% of the false readings produced using a GFIassay were identified as contradictory with in vivo data by the VCIendpoint assay. Therefore it is seen that when the in vitro VCI endpoint assay is utilized (measuring cell viability) in conjunction withthe in vitro GFI end point assay (measuring cell proliferation), thecomparative data synergistically lowers the false-positive rate for drugresponsiveness in the in vitro histoculture assay. Furthermore, whereboth the VCI and GFI in vitro indices are in agreement, in vivodrug-responsiveness testing is not generally required, or can besubstantially minimized due to the high reliability correlation from thein vitro assays.

8. Viability Assay Using Tetrazolium Metabolism

In another embodiment, the enzymatic conversion of a tetrazolium saltinto an optically detectable formazan crystal was used as a marker forviable cells.

To that end, human tumor specimens were established in histoculture asdescribed in Example 1. A panel of drugs known to have varyingeffectiveness against various types of cancer were exposed to thehistocultured tumor tissues. Drugs used were mitomycin-C (about 100ng/ml), doxorubicin (about 29 ng/ml), 5-fluorouracil (about 4 μg/ml),carmustine (about 0.2 μg/ml), cisplatin (about 1.5 μg/ml), melphalan(about 1 μg/ml), vinblastine (about 7.3 ng/ml), vincristine (about 23ng/ml) and bleomycin (about 210 ng/ml), all from Sigma Chemical Co.,(St, Louis, Mo.). The concentrations of these drugs representapproximately the levels achievable clinically and are termed "IX"concentrations. All were dissolved in physiological saline except formelphalan, which was dissolved in ethanol.

A phosphate-buffered saline solution composed of about 138.7 mM NaCl,about 2.7 mM KCl, about 8 mM Na₂ HPO₄, about 1.5 mM KH₂ PO₄ wasprepared. A quantity of3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)was dissolved in the phosphate-buffered saline to form a freshlyprepared stock solution having about 8 mg/ml MTT. The solution wasfiltered through a 0.2-μm membrane filter from Millipore, (Bedford,Mass.).

The sponge-gel-supported tumor pieces, after about 24 hour incubationwith the drugs, were transferred to drug free media and were incubatedfor about 2 hours at about 37° C. in a humidified sterile atmosphere,containing about 95% air and about 5% carbon dioxide with about 2 ml ofa new solution composed of the MTT in the saline solution at a finalconcentration of about 0.4 mg/ml.

After about 2 hours, the gels were removed from the incubation mediacontaining MTT and placed in about 2 ml of cold phosphate-bufferedsaline. The specimens were kept at about 4° C., and subsequently frozenwhereupon 4-μm frozen sections were made. Water-soluble embedding mediaTissue Tek OCT Compound from Baxter Labs (Irvine, Calif.) and a TissueTek II Cryostat, from Miles Laboratories, Inc. (Naperville, Ill.) wereused in making the frozen sections.

The slides were then dipped for about 30 s in an about 1.25 μg/mlpropidium iodide solution from Sigma, prepared in distilled water. Afterbeing dried, they were ready for pixel image analysis of formazancrystals (PIAFC) and pixel image analysis of fluorescent dye (PIAFD)measurements as described herein.

The image analysis system consisted of a Nikon Optiphot microscopeconnected to an RCA TC-1501 video camera, a Hitachi monitor and an IBMpersonal computer.

The measurements were conducted microscopically under a mercury lamp,using an IGS filter for polarized light and a DM 580 G-2A filter,composed of EX 510-560 excitation and BA 590 emission filters, forfluorescent light of the proper wavelength. The objective magnificationwas 200 X and the image was digitized by a conventional digitizer board.

The areas of brightness corresponding to the amount of formazan crystalswhich reflect polarized light or to red nuclei due to fluorescence ofthe propidium iodide fluorescent dye were calculated as the ratio of thearea of enhanced pixels to total pixels by the Image Scanner, ConwayFilter and Bright Pixel Planimeter (DS-88 Digisector Video, Confilversion 1.0 program from The Microworks, Del Mar, Calif.) running on anIBM PC-XT type computer.

The PIAFC and the PIAFD were measured in the non-drug-treated controltwice, once at the beginning of the experiment and once at the end,within about 6 hours of the beginning. During this time there were nostatistically significant changes detected, indicating that the systemwas stable during the measurement period.

The analysis of a frozen section of the colon tumor under polarizedlight after about 24 h culture in the presence of doxorubicin and about1 h incubation with MTT showed formazan crystals that were easilyobserved. The same field visualized under fluorescent light afterstaining with propidium iodide illustrated that areas of high formazancrystal formation correspond to areas that contain nuclei. The absenceof nuclei in some areas of the section corresponds to the absence offormazan crystals in those areas, thus demonstrating that MTT is beingreduced only by cells and not by the drug used with this histoculturedtissue sample. The data shows that the tumor is not significantlysensitive in vitro to the drug doxorubicin (at 29 ng/ml) after the 24 hexposure.

A different frozen section of the same tumor incubated for about 24 hwith a different drug, 1.5 μm/ml cisplatin, illustrated that the PIAFCis very low with respect to PIAFD, so that their ratio divided by thecontrol value (using the formula described above) is only 0.25%. Usingthat formula, the tumor is calculated to be 99.75% sensitive in vitro tothe drug cisplatin.

These data indicate that tetrazolium salts can be used as a metabolicmarker to detect cell viability in in vitro histoculture assays formeasuring chemosensitivity of tumor tissues to antineoplastic drugs.

The foregoing description and the examples are intended as illustrativeand are not to be taken as limiting. Still other variations within thespirit and scope of this invention are possible and will readily presentthemselves to those skilled in the art.

What is claimed is:
 1. An in vitro method for predicting the effectiveness of treating a preselected tumor tissue of a subject in vivo with an anti-tumor agent, which method comprises:a) histoculturing, in separate containers, first, second, third and fourth portions of a tumor tissue sample; b) exposing said first and second portions of said tumor tissue sample to said agent; c) determining the percentage of viable cells within said first and third portions of said histocultured tumor tissue sample to produce a viable cell index comprising a ratio of viable to non-viable cells in said first and third portions; d) determining the percentage of proliferating cells within said second and fourth portions of said histocultured tumor tissue sample to produce a growth fraction index comprising a ratio of proliferating to non-proliferating cells in said second and fourth portions; and e) wherein a reduced viable call index in said first portion as compared to said third portion combined with a reduced growth fraction index in said second portion as compared to said fourth portion substantially increases the reliability of the in vitro as say as predicting in vivo effectiveness of said antitumor agent against said tumor tissue.
 2. The method of claim 1 wherein the percentage of viable cells is determined by a process which comprises measuring the number of cells in said histocultured tissue sample that incorporate a protein synthesis marker.
 3. The method of claim 2 wherein said protein synthesis marker is ³⁵ S-methionine.
 4. The method of claim 1 wherein the percentage of viable cells is determined by a process which comprises measuring the number of cells in said histocultured tissue sample that incorporate a viable cell specific dye.
 5. The method of claim 4 wherein said viable cell specific dye is selected from the group consisting of 2'7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM); glycine-N,N'-((3',6'-dihydroxy-3-oxospiro(isobenzofuran-l(3H),9'-(9H)xanthene)-2,7-diyl)bis(methylene))bis(N-carboxymethyl)-acetoxymethyl ester (calcein-AM); 5-carboxyfluorescein diacetate (CFDA); 3,6-acridinediamine, N,N,N',N'-tetramethyl-monohydrochloride (acridine orange); 7-hydroxy-4-methylcoumarin-8-methyleneiminodiacetic acid (calcein blue); 5-oxazolecarboxylic acid 2-(6-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-(2-(2(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-methylphenoxy)ethoxy)-2-benxofuranyl)-(acetyloxy)methyl ester (fura-2AM), fluorescein diacetate and 7-hydroxy-4-carboxymethylcoumarin-8-methyleneiminodiacetic acid acetoxymethyl ester (carboxy calcein blue-AM).
 6. The method of claim 2 wherein said process further comprises measuring the number of cells in said histocultured tissue sample that incorporate a dead-cell specific dye.
 7. The method of claim 6 wherein said dead-cell specific dye is selected from the group consisting of propidium iodide, ethidium bromide and ethidium homodimer.
 8. The method of claim 1 wherein the percentage of viable cells is determined by a process which comprises measuring the number of cells in said histocultured tissue sample that metabolically convert a detectable marker.
 9. The method of claim 8 wherein said detectable marker that is metabolically converted by said viable cells is a tetrazolium salt.
 10. The method of claim 9 wherein said tetrazolium salt is 3- 4,5-dimethylthiazol-2-yl!-2,5-diphenyltetrazolium bromide (MTT).
 11. The method of claim 1 wherein the percentage of proliferating cells is determined by a process which comprises measuring the number of cells in said histocultured tissue sample that incorporate a DNA-synthesis marker.
 12. The method of claim 11 wherein said DNA-synthesis marker is selected from the group consisting of ³ H-thymidine, ³ H-deoxyadenosine, ³ H-deoxyguanosine, ³ H-deoxycytidine and ³ H-deoxyuridine.
 13. The method of claim 1 wherein the percentage of viable cells is determined by a process which comprises measuring the number of cells in said histocultured tissue sample that incorporate a fluorescent dye indicative of viability and the percentage of proliferating cells is determined by a process which comprises measuring the number of cells in said histocultured tissue sample that incorporate the DNA-synthesis marker ³ H-thymidine.
 14. A kit for in vitro prediction of the effectiveness of in vivo treatment of a tumor with an anti-minor agent, which kit comprises a viable cell-specific marker for measuring cell viability in vitro and a cell proliferation marker for measuring cell proliferation in vitro, said markers each present in an mount sufficient to perform at least one in vitro determination.
 15. The kit of claim 14 wherein said viable cell-specific marker is the fluorescent dye 2'7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and said cell proliferation marker is ³ H-thymidine.
 16. An in vitro method for predicting the effectiveness of treating a preselected tumor tissue of a subject in vivo with an antitumor agent which method comprisesdetermining the viability of tumor cells in a histocultured sample of said tumor in the presence and absence of said agent; determining the proliferation of tumor cells in a histocultured sample of said tumor tissue in the presence and absence of said agent; wherein a reduction of viability of said cells in the presence as compared to the absence of said agent combined with a reduction in proliferation of said cells in the presence as compared to the absence of said agent substantially increases the reliability of the in vitro as predicting in vivo effectiveness of said antitumor agent against said tumor tissue. 